Parkinson’s disease is one of the most common neurodegenerative disorders, and the pesticide rotenone is widely used to model Parkinson’s disease in experimental studies. Rotenone-induced mitochondrial dysfunction is associated with oxidative stress and intracellular Ca ²⁺ ([Ca ²⁺ ]ᵢ) dysregulation in neuronal systems. L-type Ca ²⁺ channels (LTCCs) contribute to Ca² ⁺ influx under oxidative stress conditions, but pharmacological inhibition of LTCCs is limited by cardiovascular side effects. Phospholipase D (PLD) has also been implicated in cellular stress-associated signaling pathways. Ginsenosides are known to influence Ca² ⁺ -related signaling; however, differences among ginsenosides in neuronal and microglial Ca² ⁺ regulation have not been fully characterized. Therefore, this study aimed to investigate the effects of ginsenosides on intracellular Ca² ⁺ homeostasis and oxidative stress of rotenone-exposed SH-SY5Y neuronal cells and BV2 microglial cells. Cell viability, superoxide dismutase (SOD) activity, interleukin-6 (IL-6) levels, intracellular Ca² ⁺ influx, and malondialdehyde (MDA) levels were evaluated. Intracellular Ca² ⁺ was measured using Fura-2 AM ratiometric fluorescence analysis. The effects of ginsenosides Rg1, Rg2, and Rd were examined using pharmacological inhibitors targeting PLD, LTCCs, and protein kinase A (PKA). Rotenone reduced cell viability and SOD activity, while increasing IL-6 levels, [Ca ²⁺ ]ᵢ influx, and MDA levels in both cell types. Treatment with ginsenosides immediately before rotenone exposure attenuated rotenone-induced increases in [Ca ²⁺ ]ᵢ and MDA levels. Ca² ⁺ responses in both SH-SY5Y and BV2 cells were sensitive to PLD inhibition. In SH-SY5Y cells, responses were additionally sensitive to LTCC inhibition, whereas BV2 cells showed weaker LTCC-associated pharmacological responses. Rd showed broader pharmacological sensitivity involving PLD-, LTCC-, and PKA-associated components, whereas Rg1 and Rg2 showed predominantly PLD-associated response patterns. These findings suggest that ginsenosides differentially modulate rotenone-associated Ca² ⁺ dysregulation through PLD-associated pharmacological pathways in a cell type-dependent manner. Overall, the present findings demonstrate distinct pharmacological response profiles among structurally different ginsenosides under rotenone-induced oxidative stress conditions.

Background: Intracellular Ca²⁺ dysregulation and neurovascular dysfunction are increasingly recognized as important mechanisms underlying neurodegenerative disorders, including Alzheimer’s disease (AD). Restoration of pathological Ca²⁺ imbalance has therefore emerged as a potential therapeutic strategy. Although ginsenoside compound K (CK) has demonstrated neuroprotective and anti-inflammatory properties, its role in Ca²⁺ homeostasis remains unclear. Methods: SH-SY5Y neuronal cells, U373 astrocytes, BV2 microglia, bEND brain endothelial cells, and MOVAS vascular smooth muscle cells were exposed to trimethyltin chloride (TMT, 5 μM, 24 h). Cell viability, real-time cell confluence, intracellular Ca²⁺ influx, and endoplasmic reticulum (ER) Ca²⁺ store release were evaluated. Ca²⁺ dynamics were analyzed using Fura-2 fluorescence, and signaling associated with CK-mediated Ca²⁺ regulation was investigated using pharmacological inhibitors. Orai1 protein expression was evaluated by Western blot analysis in SH-SY5Y and U373 cells. Results: TMT increased store-operated Ca²⁺ entry (SOCE)-mediated Ca²⁺ influx in SH-SY5Y neuronal cells without significant cytotoxicity. In contrast, TMT reduced both Ca²⁺ influx and cell viability in U373 astrocytes, BV2 microglia, and MOVAS cells, whereas bEND cells showed minimal changes. CK restored abnormal Ca²⁺ responses in a cell type-specific manner, reducing elevated Ca²⁺ influx in neuronal cells while restoring suppressed Ca²⁺ signaling in astrocytes. Consistent with these functional findings, Orai1 protein expression was increased by TMT in SH-SY5Y cells and attenuated by CK, whereas TMT reduced Orai1 expression in U373 cells, which was restored by CK. Pharmacological analyses suggested involvement of PKA- and LTCC-associated signaling in neuronal cells and PLC- and PLD-associated signaling in astrocytes. In both cell types, CK-associated Ca²⁺ responses were sensitive to NAC and the SOCE inhibitor BTP2, supporting ROS- and SOCE-associated mechanisms. Additional controls showed no significant inhibitor effects under control or CK-only conditions, strengthening the pharmacological interpretation of responses under TMT-containing conditions. Conclusions: TMT-induced Ca²⁺ dysregulation differed markedly by cell type. CK restored disrupted Ca²⁺ homeostasis in association with distinct pharmacological signaling profiles in neuronal and astrocytic cells. These findings suggest that CK may function as a cell type-specific Ca²⁺ homeostatic regulator under neurotoxic stress conditions and highlight the importance of differential Ca²⁺ regulation in neurodegenerative disease models.